In a previous study, it was demonstrated that two patients with type 1 (insulin-dependent) diabetes mellitus had autoantibodies in their serum which reacted with a 29 kDa pancreas-specific protein on two dimensional immunoblots. We have now purified and identified this pancreatic protein. The protein was purified through the use of ammonium sulfate fractionation and ion-exchange chromatography. Gel filtration chromatography established that the protein's molecular weight was doser to 25 kDa. Amino acid composition and sequence analyses demonstrated homology between the protein and chymotrypsin. Immunization of a rabbit with the protein produced an antiserum which reacted with cells in the exocrine pancreas of the rat and monkey. Preabsorption of the rabbit antiserum with either the purified protein or commercially purified chymotryspin eliminated this immunoreactivity. The rabbit antiserum also contained immunoreactivity against specific cells within the rat and monkey islets. This immunoreactivity was not preabsorbed by either the purified protein or commercially purified chymotrypsin. Possible reasons for the existence of these autoantibodies include: Insulitis with spread to exocrine tissue; chrymotrypsin contamination of insulin injections; and an increase in chymotrypsinogen production during the diabetic state.